RÉSUMÉ
Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to develop a platform technology to optimize the signal peptides for high level production of therapeutic antibodies in CHO cells. A database of signal peptides from a large number of human immunoglobulin (Ig) heavy chain (HC) and kappa light chain (LC) was generated. Most of the HC signal peptides contain 19 amino acids which can be divided into three domains and the LC signal peptides contain 22 amino acids. The signal peptides were then clustered according to sequence similarity. Based on the clustering, 8 HC and 2 LC signal peptides were analyzed for their impacts on the production of 5-top selling antibody therapeutics, namely, Herceptin, Avastin, Remicade, Rituxan, and Humira. The best HC and LC signal peptides for producing these 5 antibodies were identified. The optimized signal peptides for Rituxan is 2-fold better compared to its native signal peptides which are available in the public database. Substitution of a single amino acid in the optimized HC signal peptide for Avastin reduced its production significantly. Mass spectrometry analyses revealed that all optimized signal peptides are accurately removed in the mature antibodies. The results presented in this report are particularly important for the production of these 5 antibodies as biosimilar drugs. They also have the potential to be the best signal peptides for the production of new antibodies in CHO cells.
Sujet(s)
Anticorps monoclonaux/métabolisme , Immunoglobuline G/métabolisme , Chaines lourdes des immunoglobulines/métabolisme , Chaines légères des immunoglobulines/métabolisme , Signaux de triage des protéines/physiologie , Animaux , Cellules CHO , Cricetulus , Spectrométrie de masseRÉSUMÉ
GOLPH3 is a highly conserved protein found across the eukaryotic lineage. The yeast homolog, Vps74p, interacts with and maintains the Golgi localization of several mannosyltransferases, which is subsequently critical for N- and O-glycosylation in yeast. Through the use of a T7 phage display, we discovered a novel interaction between GOLPH3 and a mammalian glycosyltransferase, POMGnT1, which is involved in the O-mannosylation of α-dystroglycan. The cytoplasmic tail of POMGnT1 was found to be critical for mediating its interaction with GOLPH3. Loss of this interaction resulted in the inability of POMGnT1 to localize to the Golgi and reduced the functional glycosylation of α-dystroglycan. In addition, we showed that three clinically relevant mutations present in the stem domain of POMGnT1 mislocalized to the endoplasmic reticulum, highlighting the importance of identifying the molecular mechanisms responsible for Golgi localization of glycosyltransferases. Our findings reveal a novel role for GOLPH3 in mediating the Golgi localization of POMGnT1.
Sujet(s)
Appareil de Golgi/métabolisme , Protéines membranaires/métabolisme , N-acetylglucosaminyltransferase/métabolisme , Technique de Western , Dystroglycanes/métabolisme , Réticulum endoplasmique/métabolisme , Expression des gènes , Glycosylation , Cellules HEK293 , Cellules HeLa , Humains , Protéines membranaires/génétique , Microscopie de fluorescence , Mutation , N-acetylglucosaminyltransferase/génétique , Liaison aux protéines , Interférence par ARN , RT-PCRRÉSUMÉ
Nogo-A has been extensively demonstrated to play key roles in inhibiting central nervous system regeneration, regulating endoplasmic reticulum formation, and maintaining the integrity of the neuromuscular junction. In this study, an E3 ubiquitin ligase WWP1 was first identified to be a novel interacting partner for Nogo-A both in vitro and in vivo. By using CD, ITC, and NMR, we have further conducted extensive studies on all four WWP1 WW domains and their interactions with a Nogo-A peptide carrying the only PPxY motif. The results lead to several striking findings. (1) Despite containing an unstructured region, the 186-residue WWP1 fragment containing all four WW domains is able to interact with the Nogo-A(650-666) peptide with a high affinity, with a dissociation constant (K(d)) of 1.68 microM. (2) Interestingly, four isolated WW domains show differential structural properties in the free states. WW1 and WW2 are only partially folded, while WW4 is well-folded. Nevertheless, they all become well-folded upon binding to Nogo-A(650-666), with K(d) values ranging from 1.03 to 3.85 microM. (3) The solution structure of the best-folded WW4 domain is determined, and the binding-perturbed residues were derived for both WW4 and Nogo-A(650-666) by NMR HSQC titrations. Moreover, on the basis of the NMR data, the complex model is constructed by HADDOCK 2.0. This study provides rationales as well as a template Nogo-A(650-666) for further design of molecules to intervene in the WWP1-Nogo-A interaction which may regulate the Nogo-A protein level by controlling its ubiquitination.
Sujet(s)
Système nerveux central/métabolisme , Protéines de la myéline/composition chimique , Ubiquitin-protein ligases/composition chimique , Séquence d'acides aminés , Animaux , Dichroïsme circulaire , Glutathione transferase/métabolisme , Humains , Cinétique , Spectroscopie par résonance magnétique , Conformation moléculaire , Données de séquences moléculaires , Protéines de la myéline/métabolisme , Protéines Nogo , Liaison aux protéines , Similitude de séquences d'acides aminés , Ubiquitine/composition chimique , Ubiquitin-protein ligases/métabolismeRÉSUMÉ
The Na,K-ATPase gamma subunit is present primarily in kidney as two splice variants, gammaa and gammab, which differ only at their extracellular N-termini. Two distinct effects of gamma are seen in biochemical Na,K-ATPase assays of mammalian (HeLa) cells transfected with gammaa or gammab, namely, (i) a decrease in K'(ATP) probably secondary to a shift in steady-state E(1) <--> E(2) poise in favor of E(1) and (ii) an increase in cytoplasmic K(+)/Na(+) antagonism seen as an increase in K'(Na) at high K(+) concentration. Mutagenesis experiments involving alterations in extramembranous regions of gamma indicate that different regions mediate the aforementioned distinct effects and that the effects appear to be long range. Studies of ouabain-sensitive fluxes with intact cells confirm the gamma effects seen with membranes and also suggest an additional effect (increase) in apparent affinity for extracellular K(+). Alteration in gamma function was also evidenced in the behavior of a G41 -->R mutation within the transmembrane domain of gamma. G41R is associated with autosomal dominant renal magnesium wasting. Our studies show that this mutation in the gammab variant retards trafficking of gamma, but not alphabeta pumps, to the cell surface and abolishes functional effects of gamma, consistent with the conclusion that the Mg(2+) transport defect is secondary to loss of gamma modulation of Na,K-ATPase function.
Sujet(s)
Sodium-Potassium-Exchanging ATPase/composition chimique , Sodium-Potassium-Exchanging ATPase/métabolisme , Séquence d'acides aminés , Animaux , Cellules HeLa , Humains , Cinétique , Fragments peptidiques/composition chimique , Protéines recombinantes/composition chimique , Protéines recombinantes/métabolisme , TransfectionRÉSUMÉ
The two variants of the gamma subunit of the rat renal sodium pump, gamma(a) and gamma(b), have similar effects on the Na,K-ATPase. Both increase the affinity for ATP due to a shift in the enzyme's E(1) <--> E(2) conformational equilibrium toward E(1). In addition, both increase K(+) antagonism of cytoplasmic Na(+) activation. To gain insight into the structural basis for these distinct effects, extramembranous N-terminal and C-terminal mutants of gamma were expressed in rat alpha1-transfected HeLa cells. At the N terminus, the variant-distinct region was deleted (gammaNDelta7) or replaced by alanine residues (gammaN7A). At the C terminus, four (gamma(a)CDelta4) or ten (gamma(a)CDelta10) residues were deleted. None of these mutations abrogates the K(+)/Na(+) antagonism as evidenced in a similar increase in K'(Na) seen at high (100 mm) K(+) concentration. In contrast, the C-terminal as well as N-terminal deletions (gammaNDelta7, gamma(a)CDelta4, and gamma(a)CDelta10) abolished the decrease in K'(ATP) seen with wild-type gamma(a) or gamma(b). It is concluded that different regions of the gamma chain mediate the distinct functional effects of gamma, and the effects can be long-range. In the transmembrane region, the impact of G41R replacement was analyzed since this mutation is associated with autosomal dominant renal Mg(2+)-wasting in man (Meij, I. C., Koenderink, J. B., van Bokhoven, H., Assink, K. F. H., Groenestege, W. T., de Pont, J. J. H. H. M., Bindels, R. J. M., Monnens, L. A. H., Van den Heuvel, L. P. W. J., and Knoers, N. V. A. M. (2000) Nat. Genet. 26, 265-266). The results show that Gly-41 --> Arg prevents trafficking of gamma but not alphabeta pumps to the cell surface and abrogates functional effects of gamma on alphabeta pumps. These findings underscore a potentially important role of gamma in affecting solute transport, in this instance Mg(2+) reabsorption, consequent to its primary effect on the sodium pump.